title: Uhrf1-knockout E14 mESC SL_M-M-Dyad-seq Deep Sequencing Replicate B

city: Santa Barbara

first: Alex

last: Chialastri

address: 2002 BioEngineering Bldg, CNTR for Bioengineering

institute: University of California Santa Barbara

description: UHRF1 is known to recruit DNMT1 to hemimethylated DNA to enable maintenance of DNA methylation.

data_processing:
Illumina bcl2fastq2 v 2.20 software used for basecalling A custom Perl script was used to demultiplex genomic based read barcodes from RNA based read barcodes. This code is available on request. RNA enriched samples were processed for RNA transcripts, while unenriched samples were used to identify site specific 5mC as well as the status of CpG dyads as fully or hemi-methylated. For genomic site specific 5mC detection (for absolute 5mC (or 5hmC) level calling), exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 and 2 were trimmed using TrimGalore in paired end mode. The 5’ end of read 1 was clipped by 20 bases and the 3’ end of read 2 was hard clipped 34 bases after detection of the PCR amplification sequence CCACATCACCCAAACC to remove potential bias from enzymatic digestion and to avoid recounting CpG dyad detections utilized in the maintenance measurement. The 5’ end of read 2 was clipped by 9 bases to remove potential bias from the linear amplification 9-mer. The 3’ end of read 1 was also hard clipped 9 bases after detection of the Illumina adapter was performed. Each read was mapped individually using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. The bismark_methylation_extractor tool was then used to extract detected cytosines. After which custom Perl code was used to connect detected cytosines to their respective cell. CpG detection files from the same DNA strand for read 1 and read 2 were then combined and any duplicate detections where the same cytosine was read in both read 1 and read 2 were removed using the UMI. Custom codes for analyzing Dyad-seq data and the accompanying documentation is provided with this work. For genomic CpG Dyad specific calling (for 5mCpG (or 5hmC) maintenance calling), Read 1 was trimmed to 86 bases. Then exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 was trimmed using the default settings of TrimGalore. Read 1 was mapped using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. For library preparation using MspJI, a custom Perl script was used to identify 5mC positions in the genome as detected by MspJI and interrogate the methylation status of the opposing cytosine in a CpG or CpHpG dyad context from the nucleobase conversion. For library preparation using AbaSI, a custom Perl script was used to identify 5hmC positions in the genome as detected by AbaSI and interrogate the methylation status of the opposing cytosine in a CpG dyad context from the nucleobase conversion. For bulk RNA based reads, read 2 sequences were trimmed to 50 bases and mapped in the sense direction to the refseq MM10 transcriptome build supplemented with ERCC mRNA spike-in sequences using bwa v0.7.15 with parameters aln -q 0 -n 0.04 -k 2 -l 200 -t 6 -B 12. Any read mapping to multiple loci were distributed uniformly across those loci. Gene isoforms were consolidated into a gene count, and the UMI was used to deduplicate reads from each gene for each individual cell. The number of observations was converted into transcript counts based on binomial statistics as described previously by Grün et al.. Genes that were not detected in at least one cell were discarded from any downstream analysis. For single cell RNA based reads, Read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to MM10 using the gene annotation file from Ensembl . The reads were again mapped to MM10 using the transposable elements annotation file described in TEtranscripts. Transcripts with the same UMI were deduplicated, transcripts detected from genes or transposable elements were analyzed together and any that were not detected in at least one cell were removed from any downstream analysis. Required code to go from raw FASTQ files to processed data can be found on github at https://github.com/alexchialastri/scDyad-T-seq
Assembly: MM10 and hg19
Supplementary files format and content: 5mCpG Dyad maintenance detection files [noted in the naming of the file as *_CGMaintained.txt (for loci detected as maintained {e.g. fully methylated dyads for M-M-Dyad-seq}) and *_CGNonMaintained.txt (for loci detected as not maintained {e.g. hemi-methylated dyads for M-M-Dyad-seq})] list detected methylated or unmethylated CpG maintenance sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the UMI detected and column 4 is the strand of the detected mark.
Supplementary files format and content: Absolute 5mCpG detection files [noted in the naming of the file as *_BS-CpG_All.txt or *BS-CpG_All_BothStrands_Unique.txt] list detected methylated or unmethylated CpG sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the the strand of the detected mark, column 5 is the UMI detected and column 6 indicates if the cytosine was methylated (Z) or unmethylated (z) . Supplementary_files_format_and_content: For Bulk samples RNA-seq gene count files (noted in the naming of the file as *-READ_COUNTS_c.txt.gz) is a matrix with the columns indicating sample (replicate) number, and each row indicating the number of counts detected for a given gene.
Supplementary files format and content: Single cell transcript counts contain 6 files. 3 refer to the detection of unique transcripts related to genes (*_Genes_matrix.mtx.gz, *_Genes_features.tsv.gz, and *_Genes_barcodes.tsv.gz) and 3 refer to the detection of unique transcripts related to transposable elements (*_TE_matrix.mtx.gz, *_TE_features.tsv.gz, and *_TE_barcodes.tsv.gz). These set of 3 files are in the standard 10x sparse data matrix format (Market Exchange Format). The *_barcodes.tsv.gz file gives the list of cell barcodes used in the experiment. Note: these are in alphabetical order and not ordered by cell number, see supplementary file ('SingleCell_barcodesOrdered.csv') on main page for which barcodes refer to what cell number. The *_features.tsv.gz file gives the list of gene or transposable elements names. The *_matrix.mtx.gz gives the location and value of non zero elements in gene or transposable elements detection matrix, with column 1 refering to the feature, column 2 to the cell number, and column 3 to the number of transcripts detected.
Library strategy: M-M-Dyad-seq

source_name: E14 mESC

growth_protocol: All cells were maintained in incubators at 37°C and 5% CO2. Mouse embryonic stem cell line ES-E14TG2a (E14) were grown on gelatin (Millipore Sigma, ES-006-B) coated tissue culture plates with media containing high glucose DMEM (Gibco, 10569044), 1% non-essential amino acid (Gibco, 11140050), 1% Glutamax (Gibco, 35050061), 1x Penicillin-Streptomycin (Gibco, 15140122), and 15% stem cell qualified serum (Millipore Sigma, ES-009-B). The media was frozen in aliquots and used thereafter for a maximum of 2 weeks after thawing while storing it at 4°C. Once thawed, 1 µL of beta-mercaptoethanol (Gibco, 21985023), and 1 µL of LIF (Gibco, A35933) were added for every 1 mL of thawed media. Cells were washed with 1x DPBS (Gibco, 14190250) and the media was exchanged daily. Cells were routinely passaged 1:6 once they reached 75% confluency using 0.25% trypsin-EDTA (Gibco, 25200056). E14 cells grown under these conditions also describe the SL experimental group. For FACS sorting, a single-cell suspension was made using 0.25% trypsin-EDTA. The trypsin was then inactivated using serum containing medium. Afterwards, the cells were washed with 1x DPBS before being passed through a cell strainer and sorted for single cells into 384-well plates. K562 cells were grown in RPMI (Gibco, 61870036) with 10% serum (Gibco, 10437028) and 1x Penicillin-Streptomycin. When the culture reached a density of approximately 1 million cells per mL, they were split and resuspended at a density of 200,000 cells per mL. Cells were washed and FACS sorted as described for E14 cells.

extract_protocol: Bulk genomic DNA was extracted using the DNeasy kit (Qiagen, 69504) according to the manufacturer’s recommendations. Bulk total RNA was extracted using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer’s recommendations. For single cell experiments, whole cell lysate was used by soring cells via FACs directly into lysis buffer. For all bulk Dyad-seq experiments, 100 ng of purified genomic DNA was resuspended in 20 µL of glucosylation mix (1x CutSmart buffer (NEB, B7204S), 2.5x UDP-glucose, 10 U T4-BGT (NEB, M0357L)) and the samples were incubated at 37°C for 16 hours. Afterwards, 10 µL of protease mix (100 µg protease (Qiagen, 19155), 1x CutSmart buffer) was added to each sample, and the samples were heated to 50°C for 5 hours, 75°C for 20 minutes, and 80°C for 5 minutes. After this, processing of the samples differed based on the version of Dyad-seq used. For M-M-Dyad-seq and M-H-Dyad-seq, 10 µL of MspJI digestion mix (2 U MspJI, 1x enzyme activator solution, 1x CutSmart buffer) was added to each sample and the samples were heated to 37°C for 5 hours, and 65°C for 20 minutes. Next, 1 µL of barcoded 1 µM double-stranded adapter was added. Then 9 µL of ligation mix (1.11x T4 ligase reaction buffer, 4.44 mM ATP (NEB, P0756L), 2000 U T4 DNA ligase (NEB, M0202M)) was added to each sample, and the samples were incubated at 16°C for 16 hours. For H-M-Dyad-seq and H-H-Dyad-seq, 10 µL of AbaSI digestion mix (10 U AbaSI (NEB, R0665S), 1x CutSmart buffer) was added to each sample and the samples were incubated at 25°C for 2 hours, and then heated to 65°C for 20 minutes. Next, 1 µL of barcoded 1 µM double-stranded adapter was added. Then 9 µL of ligation mix (1.11x T4 ligase reaction buffer, 4.44 mM ATP (NEB, P0756L), 2000 U T4 DNA ligase (NEB, M0202M)) was added to each sample, and the samples were incubated at 16°C for 16 hours. After ligation, up to three barcoded libraries of the same type were pooled and all Dyad-seq versions were subjected to a 1x AMPure XP bead cleanup (Beckman Coulter, A63881), and eluted in 40 µL of water. M-M-Dyad-seq and H-M-Dyad-seq samples were then concentrated to a volume of 28 µL and subjected to nucleobase conversion using the NEBNext enzymatic methyl-seq conversion module (NEB, E7125S) according to the manufacturer’s recommendations except for performing the final elution step in 40 µL of water. For M-H-Dyad-seq and H-H-Dyad-seq samples, nucleobase conversion was performed using the NEBNext enzymatic methyl-seq conversion module similar to what has been described previously11. Briefly, samples were first concentrated to a volume of 17 µL. Then 4 µL of formamide (Sigma-Aldrich, F9037-100ML) was added and the samples were heated to 85°C for 10 minutes before being quenched on ice. APOBEC nucleobase conversion was performed as described by the manufacturer except for two minor changes. Samples were incubated at 37°C for 16 hours, and the final elution step was performed using 40 µL of water. To all Dyad-seq versions, the nucleobase converted samples were subjected to one round of linear amplification. To do this, 9 µL of amplification mix was added (5.56x NEBuffer 2.1 (NEB, B7202S), 2.22 mM dNTPs (NEB, N0447L), and 2.22 uM Linear amplification 9-mer: 5’- GCCTTGGCACCCGAGAATTCCANNNNNNNNN -3’) and the samples were heated to 95°C for 45 seconds before being quenched on ice. Once cold, 100 U of high concentration Klenow DNA polymerase (3’-5’ Exo-) (fisher scientific, 50-305-912) was added. Then samples were quickly vortexed, centrifuged and then incubated at 4°C for 5 minutes, followed by an increase of 1°C every 15 seconds at a ramp rate of 0.1°C per second till the samples reach 37°C which was then held for an additional 1.5 hours. Afterwards a 1.1x AMPure XP bead cleanup was performed, and the samplers were eluted in 40 µL of water before being concentrated down to 10 µL. The entire sample was then used in a linear PCR reaction by adding 15 µL of PCR mix (1.67x high-fidelity PCR mix (NEB, M0541L) and 0.67 µM Extended RPI primer: 5’- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCGGTGTAGTGGGTTTGG-3’) and performing PCR as follows: Initial denaturing at 98°C for 30 seconds, followed by 16 cycles of 98°C for 10 seconds, 59°C for 30 seconds, and 72°C for 30 seconds, and a final extension step at 72°C for 1 minute. Next, 5 µL of the linear PCR product was amplified further in a standard Illumina library PCR reaction, incorporating a uniquely indexed i7 primer. The remaining linear PCR product was stored at -20°C. To the final sequencing library, two 0.825x AMPure XP bead cleanups were performed with a final elution volume of 15 µL in water. The libraries were then quantified on an Agilent Bioanalyzer and Qubit fluorometer. Finally, libraries were subjected to paired-end 150 bp Illumina sequencing on a HiSeq platform.

treatment_protocol: Decitabine treatment: E14 mouse embryonic stem cells were cultured as described above. Upon passage of the E14 cells, SL media was supplemented with 0.05 µM of Decitabine. After 24 hours, cells were harvested using 0.25% trypsin-EDTA. The trypsin was then inactivated using serum containing medium. The cells were washed with 1x DPBS and then resuspended in 200 µL of DPBS. Genomic DNA was extracted using the DNeasy kit (Qiagen, 69504) according to the manufacturer’s recommendations. K562 cells were cultured as described above. Upon passage, the media was supplemented with 0.6 µM of Decitabine or DMSO vehicle control. After 24 hours the cells were washed and single-cell FACS sorting was performed as described above. Transition from serum to 2i and other media conditions: E14 mouse embryonic stem cells were cultured in SL conditions as described above. Upon passage, cells were resuspended in the following media depending on the condition studied. Commercial 2i media containing LIF (Millipore, SF016-200) was used for BL, G, 2i, and M experiments. For 2i, all components were used according to the manufacturer's recommendations. For G and M conditions, only the GSK3B inhibitor or MEK1/2 inhibitor was added, respectively. For the BL condition, no inhibitors were added. For the No condition, commercial 2i media without LIF (Millipore, SF002-100) was used with no inhibitors added. After 24 hours, the cells were washed with 1x DPBS and the media was exchanged. 48 hours after the initial media switch, the cells were collected using 0.25% trypsin-EDTA, quenched using serum containing media, washed in 1x DPBS and finally resuspended in 1x DPBS. The sample was then split in half for genomic DNA and total RNA extraction. In single-cell serum to 2i experiments, the same process was followed with cells being isolated into a reaction well of a 384 well plate using FACS after 3, 6, or 10 days in 2i. Cells grown in 2i were split 1:6 every other day.

value: E14 mESC


title: TraPR small RNA-seq: ago18HT-2mm-rep2

status: Public on May 14 2024

type: SRA

city: Wuhan

first: Junpeng

last: Zhan

state: Hubei

address: 1 Shizishan Street, Hongshan District

country: China

institute: Huazhong Agricultural University

library_source: transcriptomic

data_processing:
library strategy: TraPR (Trans-kingdom, rapid, affordable Purification of RNA-induced silencing complexes (RISCs)) small RNA-seq
To quantify phasiRNA abundance, sRNA-seq reads were mapped to the respective Zea genomes using Bowtie v1.3.1.Reads mapped to PHAS loci were counted using featureCounts v1.6.3 and normalized to counts per million (CPM) using edgeR v4.0.2. Differential expression analyses were performed using edgeR with a generalized linear model-based method.
Assembly: B73 RefGen_v5
Supplementary files format and content: CPM tables

molecule: total RNA

organism: Zea mays subsp. mays

source_name: anther

extract_protocol:
Fresh anthers were snap-frozen in liquid nitrogen and total RNA was extracted using the TraPR Small RNA Isolation kit (Lexogen)
sRNA-seq libraries were prepared using a Somagenics RealSeq-AC miRNA library kit following the manufacturer’s protocol.

tag: cell type

value: anther

tag: genotype

value: mostly B73

tag: group

tag: molecule subtype

value: Small RNA - Argonaute loaded


title: embryo13_cell34

city: Edinburgh

first: Lawrence

middle: E

last: Bates

address: Crewe Road

department: Human Genetics Unit

country: United Kingdom

institute: University of Edinburgh

description: lane1_Ferdinand_Embryos_Plate1_H01_AGCTCTTT

library_source: transcriptomic single cell

data_processing:
Raw reads were processed as previously (https://doi.org/10.1038/s41467-018-03149-4), aligning to the GRCh38 reference genome using Hisat2 (https://doi.org/10.1038/s41587-019-0201-4).
Gene expression counts were computed from the mapped data using the featureCounts function within RSubread (https://doi.org/10.1093/nar/gkz114).
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text file with raw counts for each sample

molecule: polyA RNA

organism: Homo sapiens

source_name: Blastocyst

extract_protocol:
Cells were collected directly into 2.5 μl methyltransferase reaction mixture which was comprised of 1 × M.CviPI Reaction buffer, 2 U M.CviPI, 160 μM S-adenosylmethionine, 1 U μl−1 RNAsein, 0.1% IGEPAL then incubated for 15 min at 37 °C. The reaction was stopped and the RNA preserved with the addition of 5 μl RLT plus.
Single-cell RNA sequencing libraries were prepared according to the RNA half of the scNMT-seq protocol (https://doi.org/10.1038/s41467-018-03149-4). Briefly, biotinylated oligo-dT was bound to magnetic beads which were added to single-cell lysates in 96w plates. The captured mRNA was then reverse transcribed and PCR amplified on beads accordingto the Smartseq2 protocol (https://doi.org/10.1038/nprot.2016.138,https://doi.org/10.1038/nprot.2014.006). Indexed Illumina libraries were prepared from the amplified cDNA using Nextera XT reagents and then pooled and sequenced on a HiSeq 2000 instrument using 75bp paired-end reads.

tag: tissue

value: Blastocyst

value: ICM

value: WT

tag: developmental stage

tag: Sex

value: Female


title: embryo13_cell29

description: lane1_Ferdinand_Embryos_Plate1_F04_CTTCTAGA


title: Brain stem, MK-801, rep6

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

data_processing:
FASTQ-file generation and demultiplexing using bcl-convert v. 4.0.3
Align to genome and determine transcript and gene counts with the nfcore-rnaseq pipeline v. 3.10.1 using the STAR->Salmon workflow.
Assembly: GRCm38
Supplementary files format and content: Tab-seperated file containing the raw gene counts for each sample. Rows specify genes by ENSEMBL_ID, and columns specify the sample
Supplementary files format and content: Tab-seperated file containing the raw transcript counts for each sample. Rows specify genes by ENSEMBL_ID, and columns specify the sample

organism: Mus musculus

source_name: Brain stem

extract_protocol:
RNA was isolated using the RNeasy mini kit (Qiagen, US) according to the manufacturer’s protocol.
Libraries were prepared using the Universal Plus mRNA-seq protocol (Tecan) as recommended by the manufacturer. Libraries were quantified with NuQuant, quality checked using a TapeStation instrument (Agilent Technologies) and subjected to 52-bp paired-end sequencing on a NovaSeq 6000.

value: Brain stem

tag: treatment


title: Uhrf1-knockout E14 mESC SL_M-M-Dyad-seq Deep Sequencing Replicate A

state: CA

country: USA

library_source: genomic

molecule: genomic DNA

tag: cell line


title: Brain stem, MK-801, rep5


title: E14 mESC SL_M-M-Dyad-seq Deep Sequencing Replicate B

description: Follow up experiment: Deep sequencing for sequencing library statistics


title: embryo13_cell28

description: lane1_Ferdinand_Embryos_Plate1_F03_CTCGTCGA


title: Brain stem, MK-801, rep4


title: TraPR small RNA-seq: ago18HT-2mm-rep1


title: TraPR small RNA-seq: ago18HM-5mm-rep3


title: embryo13_cell27

description: lane1_Ferdinand_Embryos_Plate1_F02_GGCTGCTT


title: E14 mESC SL_M-M-Dyad-seq Deep Sequencing Replicate A


title: Brain stem, MK-801, rep3


title: E14 mESC SL_M-H-Dyad-seq Deep Sequencing

data_processing:
Illumina bcl2fastq2 v 2.20 software used for basecalling A custom Perl script was used to demultiplex genomic based read barcodes from RNA based read barcodes. This code is available on request. RNA enriched samples were processed for RNA transcripts, while unenriched samples were used to identify site specific 5mC as well as the status of CpG dyads as fully or hemi-methylated. For genomic site specific 5mC detection (for absolute 5mC (or 5hmC) level calling), exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 and 2 were trimmed using TrimGalore in paired end mode. The 5’ end of read 1 was clipped by 20 bases and the 3’ end of read 2 was hard clipped 34 bases after detection of the PCR amplification sequence CCACATCACCCAAACC to remove potential bias from enzymatic digestion and to avoid recounting CpG dyad detections utilized in the maintenance measurement. The 5’ end of read 2 was clipped by 9 bases to remove potential bias from the linear amplification 9-mer. The 3’ end of read 1 was also hard clipped 9 bases after detection of the Illumina adapter was performed. Each read was mapped individually using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. The bismark_methylation_extractor tool was then used to extract detected cytosines. After which custom Perl code was used to connect detected cytosines to their respective cell. CpG detection files from the same DNA strand for read 1 and read 2 were then combined and any duplicate detections where the same cytosine was read in both read 1 and read 2 were removed using the UMI. Custom codes for analyzing Dyad-seq data and the accompanying documentation is provided with this work. For genomic CpG Dyad specific calling (for 5mCpG (or 5hmC) maintenance calling), Read 1 was trimmed to 86 bases. Then exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 was trimmed using the default settings of TrimGalore. Read 1 was mapped using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. For library preparation using MspJI, a custom Perl script was used to identify 5mC positions in the genome as detected by MspJI and interrogate the methylation status of the opposing cytosine in a CpG or CpHpG dyad context from the nucleobase conversion. For library preparation using AbaSI, a custom Perl script was used to identify 5hmC positions in the genome as detected by AbaSI and interrogate the methylation status of the opposing cytosine in a CpG dyad context from the nucleobase conversion. For bulk RNA based reads, read 2 sequences were trimmed to 50 bases and mapped in the sense direction to the refseq MM10 transcriptome build supplemented with ERCC mRNA spike-in sequences using bwa v0.7.15 with parameters aln -q 0 -n 0.04 -k 2 -l 200 -t 6 -B 12. Any read mapping to multiple loci were distributed uniformly across those loci. Gene isoforms were consolidated into a gene count, and the UMI was used to deduplicate reads from each gene for each individual cell. The number of observations was converted into transcript counts based on binomial statistics as described previously by Grün et al.. Genes that were not detected in at least one cell were discarded from any downstream analysis. For single cell RNA based reads, Read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to MM10 using the gene annotation file from Ensembl . The reads were again mapped to MM10 using the transposable elements annotation file described in TEtranscripts. Transcripts with the same UMI were deduplicated, transcripts detected from genes or transposable elements were analyzed together and any that were not detected in at least one cell were removed from any downstream analysis. Required code to go from raw FASTQ files to processed data can be found on github at https://github.com/alexchialastri/scDyad-T-seq
Assembly: MM10 and hg19
Supplementary files format and content: 5mCpG Dyad maintenance detection files [noted in the naming of the file as *_CGMaintained.txt (for loci detected as maintained {e.g. fully methylated dyads for M-M-Dyad-seq}) and *_CGNonMaintained.txt (for loci detected as not maintained {e.g. hemi-methylated dyads for M-M-Dyad-seq})] list detected methylated or unmethylated CpG maintenance sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the UMI detected and column 4 is the strand of the detected mark.
Supplementary files format and content: Absolute 5mCpG detection files [noted in the naming of the file as *_BS-CpG_All.txt or *BS-CpG_All_BothStrands_Unique.txt] list detected methylated or unmethylated CpG sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the the strand of the detected mark, column 5 is the UMI detected and column 6 indicates if the cytosine was methylated (Z) or unmethylated (z) . Supplementary_files_format_and_content: For Bulk samples RNA-seq gene count files (noted in the naming of the file as *-READ_COUNTS_c.txt.gz) is a matrix with the columns indicating sample (replicate) number, and each row indicating the number of counts detected for a given gene.
Supplementary files format and content: Single cell transcript counts contain 6 files. 3 refer to the detection of unique transcripts related to genes (*_Genes_matrix.mtx.gz, *_Genes_features.tsv.gz, and *_Genes_barcodes.tsv.gz) and 3 refer to the detection of unique transcripts related to transposable elements (*_TE_matrix.mtx.gz, *_TE_features.tsv.gz, and *_TE_barcodes.tsv.gz). These set of 3 files are in the standard 10x sparse data matrix format (Market Exchange Format). The *_barcodes.tsv.gz file gives the list of cell barcodes used in the experiment. Note: these are in alphabetical order and not ordered by cell number, see supplementary file ('SingleCell_barcodesOrdered.csv') on main page for which barcodes refer to what cell number. The *_features.tsv.gz file gives the list of gene or transposable elements names. The *_matrix.mtx.gz gives the location and value of non zero elements in gene or transposable elements detection matrix, with column 1 refering to the feature, column 2 to the cell number, and column 3 to the number of transcripts detected.
Library strategy: M-H-Dyad-seq


title: TraPR small RNA-seq: ago18HM-5mm-rep2


title: DMSO-treated_MRC5_rep1

city: Houston

first: Hong Seok

last: Shim

state: TX

institute: MD Anderson Cancer Center

data_processing:
The NextSeq RTA (version 2.4.11.0) was used for base calling. bcl2fastq (version 2.20.0) was used to convert the .bcl files into .fastq files. Fastq files were aligned to the human reference genome (GRCh38) using HISAT2 software and assembled using StringTie. R package DESeq2 was used for data normalization and differential expression analysis.
Assembly: GRCh38
Supplementary files format and content: raw counts

source_name: DMSO-treated human MRC5 cells

growth_protocol: Fibroblasts were cultured in EMEM with 10% Fetal Bovine Serum (FBS), 1X penicillin/streptomycin (Invitrogen).

extract_protocol:
RNA was harvested using TRIzol Reagent (Invitrogen). 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: Fibroblasts were treated with DMSO or TAC.

value: human MRC5 cells

value: DMSO-treated


title: embryo13_cell26

description: lane1_Ferdinand_Embryos_Plate1_F01_TCGTGGTT


title: Brain stem, MK-801, rep2


title: embryo13_cell25

description: lane1_Ferdinand_Embryos_Plate1_E05_GTCACGTA


title: Brain stem, MK-801, rep1


title: E14 mESC SL_H-M-Dyad-seq Deep Sequencing

data_processing:
Illumina bcl2fastq2 v 2.20 software used for basecalling A custom Perl script was used to demultiplex genomic based read barcodes from RNA based read barcodes. This code is available on request. RNA enriched samples were processed for RNA transcripts, while unenriched samples were used to identify site specific 5mC as well as the status of CpG dyads as fully or hemi-methylated. For genomic site specific 5mC detection (for absolute 5mC (or 5hmC) level calling), exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 and 2 were trimmed using TrimGalore in paired end mode. The 5’ end of read 1 was clipped by 20 bases and the 3’ end of read 2 was hard clipped 34 bases after detection of the PCR amplification sequence CCACATCACCCAAACC to remove potential bias from enzymatic digestion and to avoid recounting CpG dyad detections utilized in the maintenance measurement. The 5’ end of read 2 was clipped by 9 bases to remove potential bias from the linear amplification 9-mer. The 3’ end of read 1 was also hard clipped 9 bases after detection of the Illumina adapter was performed. Each read was mapped individually using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. The bismark_methylation_extractor tool was then used to extract detected cytosines. After which custom Perl code was used to connect detected cytosines to their respective cell. CpG detection files from the same DNA strand for read 1 and read 2 were then combined and any duplicate detections where the same cytosine was read in both read 1 and read 2 were removed using the UMI. Custom codes for analyzing Dyad-seq data and the accompanying documentation is provided with this work. For genomic CpG Dyad specific calling (for 5mCpG (or 5hmC) maintenance calling), Read 1 was trimmed to 86 bases. Then exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 was trimmed using the default settings of TrimGalore. Read 1 was mapped using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. For library preparation using MspJI, a custom Perl script was used to identify 5mC positions in the genome as detected by MspJI and interrogate the methylation status of the opposing cytosine in a CpG or CpHpG dyad context from the nucleobase conversion. For library preparation using AbaSI, a custom Perl script was used to identify 5hmC positions in the genome as detected by AbaSI and interrogate the methylation status of the opposing cytosine in a CpG dyad context from the nucleobase conversion. For bulk RNA based reads, read 2 sequences were trimmed to 50 bases and mapped in the sense direction to the refseq MM10 transcriptome build supplemented with ERCC mRNA spike-in sequences using bwa v0.7.15 with parameters aln -q 0 -n 0.04 -k 2 -l 200 -t 6 -B 12. Any read mapping to multiple loci were distributed uniformly across those loci. Gene isoforms were consolidated into a gene count, and the UMI was used to deduplicate reads from each gene for each individual cell. The number of observations was converted into transcript counts based on binomial statistics as described previously by Grün et al.. Genes that were not detected in at least one cell were discarded from any downstream analysis. For single cell RNA based reads, Read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to MM10 using the gene annotation file from Ensembl . The reads were again mapped to MM10 using the transposable elements annotation file described in TEtranscripts. Transcripts with the same UMI were deduplicated, transcripts detected from genes or transposable elements were analyzed together and any that were not detected in at least one cell were removed from any downstream analysis. Required code to go from raw FASTQ files to processed data can be found on github at https://github.com/alexchialastri/scDyad-T-seq
Assembly: MM10 and hg19
Supplementary files format and content: 5mCpG Dyad maintenance detection files [noted in the naming of the file as *_CGMaintained.txt (for loci detected as maintained {e.g. fully methylated dyads for M-M-Dyad-seq}) and *_CGNonMaintained.txt (for loci detected as not maintained {e.g. hemi-methylated dyads for M-M-Dyad-seq})] list detected methylated or unmethylated CpG maintenance sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the UMI detected and column 4 is the strand of the detected mark.
Supplementary files format and content: Absolute 5mCpG detection files [noted in the naming of the file as *_BS-CpG_All.txt or *BS-CpG_All_BothStrands_Unique.txt] list detected methylated or unmethylated CpG sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the the strand of the detected mark, column 5 is the UMI detected and column 6 indicates if the cytosine was methylated (Z) or unmethylated (z) . Supplementary_files_format_and_content: For Bulk samples RNA-seq gene count files (noted in the naming of the file as *-READ_COUNTS_c.txt.gz) is a matrix with the columns indicating sample (replicate) number, and each row indicating the number of counts detected for a given gene.
Supplementary files format and content: Single cell transcript counts contain 6 files. 3 refer to the detection of unique transcripts related to genes (*_Genes_matrix.mtx.gz, *_Genes_features.tsv.gz, and *_Genes_barcodes.tsv.gz) and 3 refer to the detection of unique transcripts related to transposable elements (*_TE_matrix.mtx.gz, *_TE_features.tsv.gz, and *_TE_barcodes.tsv.gz). These set of 3 files are in the standard 10x sparse data matrix format (Market Exchange Format). The *_barcodes.tsv.gz file gives the list of cell barcodes used in the experiment. Note: these are in alphabetical order and not ordered by cell number, see supplementary file ('SingleCell_barcodesOrdered.csv') on main page for which barcodes refer to what cell number. The *_features.tsv.gz file gives the list of gene or transposable elements names. The *_matrix.mtx.gz gives the location and value of non zero elements in gene or transposable elements detection matrix, with column 1 refering to the feature, column 2 to the cell number, and column 3 to the number of transcripts detected.
Library strategy: H-M-Dyad-seq


title: embryo13_cell24

description: lane1_Ferdinand_Embryos_Plate1_E04_TCTGACGC


title: TraPR small RNA-seq: ago18HM-5mm-rep1


title: TraPR small RNA-seq: ago18HM-2mm-rep2


title: E14 mESC SL_H-H-Dyad-seq Deep Sequencing

data_processing:
Illumina bcl2fastq2 v 2.20 software used for basecalling A custom Perl script was used to demultiplex genomic based read barcodes from RNA based read barcodes. This code is available on request. RNA enriched samples were processed for RNA transcripts, while unenriched samples were used to identify site specific 5mC as well as the status of CpG dyads as fully or hemi-methylated. For genomic site specific 5mC detection (for absolute 5mC (or 5hmC) level calling), exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 and 2 were trimmed using TrimGalore in paired end mode. The 5’ end of read 1 was clipped by 20 bases and the 3’ end of read 2 was hard clipped 34 bases after detection of the PCR amplification sequence CCACATCACCCAAACC to remove potential bias from enzymatic digestion and to avoid recounting CpG dyad detections utilized in the maintenance measurement. The 5’ end of read 2 was clipped by 9 bases to remove potential bias from the linear amplification 9-mer. The 3’ end of read 1 was also hard clipped 9 bases after detection of the Illumina adapter was performed. Each read was mapped individually using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. The bismark_methylation_extractor tool was then used to extract detected cytosines. After which custom Perl code was used to connect detected cytosines to their respective cell. CpG detection files from the same DNA strand for read 1 and read 2 were then combined and any duplicate detections where the same cytosine was read in both read 1 and read 2 were removed using the UMI. Custom codes for analyzing Dyad-seq data and the accompanying documentation is provided with this work. For genomic CpG Dyad specific calling (for 5mCpG (or 5hmC) maintenance calling), Read 1 was trimmed to 86 bases. Then exact duplicates were removed using Clumpify from BBTools. A custom Perl script was use to extract the single cell barcode and UMI from each read. Then read 1 was trimmed using the default settings of TrimGalore. Read 1 was mapped using Bismark v0.21.0 in conjunction with Bowtie2 v2.3.5 to MM10 or hg19 for mouse or human samples, respectively. Using Bismark, both resulting sam files were deduplicated further using UMI, cell barcode and mapping location. For library preparation using MspJI, a custom Perl script was used to identify 5mC positions in the genome as detected by MspJI and interrogate the methylation status of the opposing cytosine in a CpG or CpHpG dyad context from the nucleobase conversion. For library preparation using AbaSI, a custom Perl script was used to identify 5hmC positions in the genome as detected by AbaSI and interrogate the methylation status of the opposing cytosine in a CpG dyad context from the nucleobase conversion. For bulk RNA based reads, read 2 sequences were trimmed to 50 bases and mapped in the sense direction to the refseq MM10 transcriptome build supplemented with ERCC mRNA spike-in sequences using bwa v0.7.15 with parameters aln -q 0 -n 0.04 -k 2 -l 200 -t 6 -B 12. Any read mapping to multiple loci were distributed uniformly across those loci. Gene isoforms were consolidated into a gene count, and the UMI was used to deduplicate reads from each gene for each individual cell. The number of observations was converted into transcript counts based on binomial statistics as described previously by Grün et al.. Genes that were not detected in at least one cell were discarded from any downstream analysis. For single cell RNA based reads, Read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to MM10 using the gene annotation file from Ensembl . The reads were again mapped to MM10 using the transposable elements annotation file described in TEtranscripts. Transcripts with the same UMI were deduplicated, transcripts detected from genes or transposable elements were analyzed together and any that were not detected in at least one cell were removed from any downstream analysis. Required code to go from raw FASTQ files to processed data can be found on github at https://github.com/alexchialastri/scDyad-T-seq
Assembly: MM10 and hg19
Supplementary files format and content: 5mCpG Dyad maintenance detection files [noted in the naming of the file as *_CGMaintained.txt (for loci detected as maintained {e.g. fully methylated dyads for M-M-Dyad-seq}) and *_CGNonMaintained.txt (for loci detected as not maintained {e.g. hemi-methylated dyads for M-M-Dyad-seq})] list detected methylated or unmethylated CpG maintenance sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the UMI detected and column 4 is the strand of the detected mark.
Supplementary files format and content: Absolute 5mCpG detection files [noted in the naming of the file as *_BS-CpG_All.txt or *BS-CpG_All_BothStrands_Unique.txt] list detected methylated or unmethylated CpG sites. Column 1 is the cell number (in bulk this is the sample replicate indicator), column 2 is the chromosome (with chr23 indicating the X chromosome, chr24 indicating the Y chromosome, and chr25 indicating a mitocondrial based detection), column 3 is the genomic loci, column 4 is the the strand of the detected mark, column 5 is the UMI detected and column 6 indicates if the cytosine was methylated (Z) or unmethylated (z) . Supplementary_files_format_and_content: For Bulk samples RNA-seq gene count files (noted in the naming of the file as *-READ_COUNTS_c.txt.gz) is a matrix with the columns indicating sample (replicate) number, and each row indicating the number of counts detected for a given gene.
Supplementary files format and content: Single cell transcript counts contain 6 files. 3 refer to the detection of unique transcripts related to genes (*_Genes_matrix.mtx.gz, *_Genes_features.tsv.gz, and *_Genes_barcodes.tsv.gz) and 3 refer to the detection of unique transcripts related to transposable elements (*_TE_matrix.mtx.gz, *_TE_features.tsv.gz, and *_TE_barcodes.tsv.gz). These set of 3 files are in the standard 10x sparse data matrix format (Market Exchange Format). The *_barcodes.tsv.gz file gives the list of cell barcodes used in the experiment. Note: these are in alphabetical order and not ordered by cell number, see supplementary file ('SingleCell_barcodesOrdered.csv') on main page for which barcodes refer to what cell number. The *_features.tsv.gz file gives the list of gene or transposable elements names. The *_matrix.mtx.gz gives the location and value of non zero elements in gene or transposable elements detection matrix, with column 1 refering to the feature, column 2 to the cell number, and column 3 to the number of transcripts detected.
Library strategy: H-H-Dyad-seq


title: TraPR small RNA-seq: ago18HM-2mm-rep1


title: Brain stem, Vehicle, rep7

value: Vehicle


title: nanoPARE: ago18HT-5mm-rep3

description:
cleavage_sites_miRNA_ago18HT_5mm_3.tsv.gz
cleavage_sites_phasiRNA_ago18HT_5mm_3.tsv.gz

data_processing:
Small RNA-transcript interactions in each anther sample were identified using the previously published nanoPARE analysis pipeline.
Assembly: Respective reference genomes of Zea varieties
Supplementary files format and content: Tables of cleavage sites

extract_protocol:
Fresh anthers were snap-frozen in liquid nitrogen and total RNA was extracted using the TRI reagent (Sigma-Aldrich).
RNA-seq (Smart-seq2) and nanoPARE libraries were prepared from 5 ng total RNA per sample using the nanoPARE library preparation protocol.

value: polyA RNA - 5' end


title: S10_Veh

city: Melbourne

first: Wah Chin

last: Boon

state: Victoria

address: 30 Royal Parade Parkville

country: Australia

institute: The Florey Institute of Neuroscience and Mental Health

data_processing:
The libraries were sequenced with 50bp single end reads using an Illumina Hiseq and read quality assessed using FastQC
Untrimmed reads were aligned to mouse mm10 genome using Subjunc aligner (version 1.4.4) within the Subread package
Sequencing data was then summarized into reads per transcript using Feature counts and the Gencode gene models for the mouse GRCm38/mm10 genome build (August 2014 freeze)
Normalisation and statistical analysis on the count data was executed using EdgeR (version edgeR_3.4.2 in R studio, R version 3.0.2) after removing features with less than 10 counts per million for at least 3 of the samples
The data was scaled using trimmed mean of M-values (TMM)
Assembly: mouse GRCm38/mm10
Supplementary files format and content: csv file containing raw counts for each sample

source_name: brain (primary cortical cell culture)

growth_protocol: Cells were seeded in 24-well plates containing 12 mm glass coverslips, coated with 100 µg/mL poly D-lysine to a density of ~0.45 x 106 cells/well and incubated in a humidified CO2 incubator (5% CO2, 37°C).

extract_protocol:
Total RNA was extracted using PARIS kit (Invitrogen) according to protocol supplied by the manufacturer
cDNA libraries were generated using the SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing kit (Agilent Technologies), according to manufacturer’s instructions.

treatment_protocol: DSMO or 1mM 10HDA

tag: strain

value: brain (primary cortical cell culture)

tag: age

value: gestational day 15.5

value: SRY+

tag: culture

value: DSMO


title: Brain stem, Vehicle, rep6


title: embryo13_cell22

description: lane1_Ferdinand_Embryos_Plate1_E02_TGTCATCT


title: embryo13_cell23

description: lane1_Ferdinand_Embryos_Plate1_E03_AGTCCAAC


title: nanoPARE: ago18HT-5mm-rep2

description:
cleavage_sites_miRNA_ago18HT_5mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HT_5mm_2.tsv.gz


title: Brain stem, Vehicle, rep5


title: S9_10HDA


title: S6_Veh


title: nanoPARE: ago18HT-5mm-rep1

description:
cleavage_sites_miRNA_ago18HT_5mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HT_5mm_1.tsv.gz


title: embryo13_cell20

description: lane1_Ferdinand_Embryos_Plate1_D05_ATCAACCG


title: Brain stem, Vehicle, rep4


title: embryo13_cell21

description: lane1_Ferdinand_Embryos_Plate1_E01_TACCGCAA


title: S5_10HDA


title: Brain stem, Vehicle, rep3


title: Brain stem, Vehicle, rep8


title: S2_Veh


title: embryo13_cell18

description: lane1_Ferdinand_Embryos_Plate1_D03_AACGTGTG


title: nanoPARE: ago18HT-2mm-rep2

description:
cleavage_sites_miRNA_ago18HT_2mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HT_2mm_2.tsv.gz


title: Brain stem, Vehicle, rep2


title: embryo13_cell19

description: lane1_Ferdinand_Embryos_Plate1_D04_ACCTCTGG


title: S1_10HDA


title: nanoPARE: ago18HT-2mm-rep3

description:
cleavage_sites_miRNA_ago18HT_2mm_3.tsv.gz
cleavage_sites_phasiRNA_ago18HT_2mm_3.tsv.gz


title: nanoPARE: ago18HT-0.5mm-rep2

description:
cleavage_sites_miRNA_ago18HT_0.5mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HT_0.5mm_2.tsv.gz


title: Brain stem, Vehicle, rep1


title: embryo13_cell17

description: lane1_Ferdinand_Embryos_Plate1_D02_AGGCAGCT


title: embryo13_cell16

description: lane1_Ferdinand_Embryos_Plate1_D01_CGGAGGAA


title: embryo13_cell15

description: lane1_Ferdinand_Embryos_Plate1_C05_GCTATGGA


title: nanoPARE: ago18HT-0.5mm-rep1

description:
cleavage_sites_miRNA_ago18HT_0.5mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HT_0.5mm_1.tsv.gz


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_12

city: PHILADELPHIA

first: Avital

last: Swisa

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

description:
matrix column name: Foxa1/2-rep6-D4134
D4134_RNAseq

data_processing:
Basecalls performed using NextSeq Control Software 2.2.0.4, RTA 2.4.11 or HiSeq Control Software HD 3.4.0.38, RTA 2.7.7 (for distal colon) and NovaSeq Control Software v1.5 (for proximal)
FASTQ files generated using bcl2fastq v2.20.0.422  (for proximal)
The adapter trimming was performed using fastq-mcf program (v1.05) and cutadapt (v2.5).
Kallisto (v0.46.2) was used to pseudoalign samples to the GRCm38 mouse transcriptome without bootstraps.
Kallisto count tables were read into edgeR via TxImport, genes with less than 1 cpm across all samples in one group were removed, and differential gene expression analysis was performed between cell types.
Assembly: mm10
Supplementary files format and content: tsv files with normalized abundance (TPM) for each sample generated by Kallisto and csv matrix table with CPM generated with edgeR

source_name: distal colon

extract_protocol:
RNA was extracted from 1 cm segments of colonic tissues using TRIzol reagent (Invitrogen, 15596026) and RNeasy Micro Kit (Qiagen, 74004).
Libraries were made with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530), using the Poly(A) mRNA Magnetic Isolation Module (NEB, E7490)

value: distal colon

value: Mixed background

value: Villin1-Cre; Foxa1lox/lox; Foxa2lox/lox

value: Male

value: 11 months


title: SS HTN

city: Bryan

first: Brett

last: Mitchell

address: 8441 Riverside Pkwy

department: Medical Physiology

institute: Texas A&M University

description: 10x Genomics (standard methods)

data_processing:
The resulting FASTQ files were aligned with Cell Ranger 7.0.0 via 10X Genomics Cloud Analysis using the mm10 (2020 revision) mouse genome assembly.
The files for each group were loaded into Seurat version 4.1. Cells with <300 or >3000 genes and cells with a mitochondrial gene ratio above 10% were removed and genes expressed by <50 cells and mitochondrial genes were removed. Additionally, DoubletFinder 2.0.3 was run to exclude likely doublets in the sample. Counts were normalized and scaled.
Due to the low number of target cell type (LECs), the control samples were merged and used as a shared control for each model, so they were not utilized separately. To better determine model agnostic changes, we also utilized a merged HTN group (in addition to the individual models).
Assembly: mm10
Supplementary files format and content: Seurat-derived cell by gene matrix of normalized and scaled counts in comma-separated files

source_name: Kidney

extract_protocol:
Mice were euthanized and the kidneys from all mice in each group (A2 HTN, A2 Ctrl, SS HTN, SS Ctrl) were pooled, minced, and subjected to enzymatic and mechanical dissociation. The cells were filtered, washed, and then incubated with biotinylated anti-CD31 antibodies. CD31+ cells were selected for using the Thermofisher CELLection Biotin Binder Kit (catalog number: 11533D) using its standard protocol and the process was repeated using biotinylated anti-podoplanin antibodies.
The library was prepared using the Chromium Next GEM Single Cell 3’ Reagent Kit V3.1 and its standard protocol.

value: Kidney

value: 10-14 weeks

value: salt sensitive HTN


title: SS Ctrl

description: 10x Genomics (standard methods)

value: salt sensitive control (normal diet)


title: embryo13_cell14

description: lane1_Ferdinand_Embryos_Plate1_C04_GTGCAGTA

value: Blastocyst

value: ICM

value: WT

value: Female


title: nanoPARE: ago18HM-5mm-rep2

description:
cleavage_sites_miRNA_ago18HM_5mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HM_5mm_2.tsv.gz

value: anther

value: mostly B73

value: polyA RNA - 5' end


title: nanoPARE: ago18HM-5mm-rep3

description:
cleavage_sites_miRNA_ago18HM_5mm_3.tsv.gz
cleavage_sites_phasiRNA_ago18HM_5mm_3.tsv.gz


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_11

description:
matrix column name: Foxa1/2-rep5-D3982
D3982_RNAseq

value: 13 months


title: embryo13_cell13

description: lane1_Ferdinand_Embryos_Plate1_C03_GGCACAAC


title: nanoPARE: ago18HM-5mm-rep1

description:
cleavage_sites_miRNA_ago18HM_5mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HM_5mm_1.tsv.gz


title: embryo13_cell12

description: lane1_Ferdinand_Embryos_Plate1_C02_GCGATTAC


title: nanoPARE: ago18HM-2mm-rep3

description:
cleavage_sites_miRNA_ago18HM_2mm_3.tsv.gz
cleavage_sites_phasiRNA_ago18HM_2mm_3.tsv.gz


title: nanoPARE: ago18HT-2mm-rep1

description:
cleavage_sites_miRNA_ago18HT_2mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HT_2mm_1.tsv.gz


title: 1098-6 SMAD4- SB 3

city: Nashville

first: Anna

last: Mean

state: Tennessee

address: 1161 21st Ave S

department: Department of Surgery

institute: Vanderbilt University Medical Center

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Cutadapt (cutadapt -j 2 -q 20 -a AGATCGGAAGAGCACACGTC -A AGATCGGAAGAGCGTCGTGT -m 30 -n 3).
Trimmed sequence reads were mapped to GrCM38/mm10 using START.
Read count extraction was performed using featureCounts.
Read count normalization was performed using DESeq2.
Assembly: mm10
Supplementary files format and content: tab-delimited csv files include raw counts for each Sample.

source_name: Papillomas

growth_protocol: Organoids were maintained in advanced DMEM/F12, 50 ng/ml EGF, 25 ng/ml FGF10, 1X N2, 1X B27 with 40% WNT3A conditioned medium, 10% R-Spondin conditioned medium, 10% Noggin conditioned medium, and antibiotics.

extract_protocol:
RNA was harvested using RNeasy kit (Qiagen). 100–300 ng  of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared by Vanderbilt Technologies for Advanced Genomics (VANTAGE).

treatment_protocol: Organoids were treated with SB431542 (10uM) (Sigma Aldrich, St. Louis, MO), LDN193189 (5nM) (Sigma Aldrich), rhTGFb1 (3ng/ml) (R&D Systems, Minneapolis, MN), rhBMP2 (20ng/ml) (R&D Systems). Vehicle controls were: DMSO (SB431542), water (LDN193189), 4mM HCl, 0.1% BSA in PBS (TGFb1, BMP2).

value: Papillomas


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_8

description:
matrix column name: Foxa1/2-rep2-D3855
D3855_RNAseq

value: distal colon

value: Mixed background

value: 9 months


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_9

description:
matrix column name: Foxa1/2-rep3-D3919
D3919_RNAseq

value: Villin1-Cre; Foxa1lox/lox; Foxa2lox/lox

value: Male

value: 10 months


title: 1097-6 SMAD4- SB 2


title: nanoPARE: ago18HM-2mm-rep2

description:
cleavage_sites_miRNA_ago18HM_2mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HM_2mm_2.tsv.gz


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_13

description:
matrix column name: Foxa1/2-rep7-D4799
D4799_RNAseq

value: 12 months


title: embryo13_cell11

description: lane1_Ferdinand_Embryos_Plate1_C01_GATGGTCC


title: 1095-6 SMAD4- SB 1


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_6

description:
matrix column name: Control-rep6-D4635
D4635_RNAseq

value: control

value: Villin1-Cre; Foxa1lox/lox; Foxa2lox/wt

value: 18 months


title: embryo13_cell9

description: lane1_Ferdinand_Embryos_Plate1_B04_TTGCGTAC

value: Blastocyst

value: ICM

value: WT


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_7

description:
matrix column name: Foxa1/2-rep1-D3854
D3854_RNAseq


title: nanoPARE: ago18HM-2mm-rep1

description:
cleavage_sites_miRNA_ago18HM_2mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HM_2mm_1.tsv.gz

value: anther

value: mostly B73


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_5

description:
matrix column name: Control-Rep5-D4634
D4634_RNAseq

value: Foxa1lox/lox; Foxa2lox/lox


title: 1098-5 SMAD4- veh 3

value: Papillomas

value: DMSO


title: nanoPARE: ago18HM-0.5mm-rep3

description:
cleavage_sites_miRNA_ago18HM_0.5mm_3.tsv.gz
cleavage_sites_phasiRNA_ago18HM_0.5mm_3.tsv.gz

value: polyA RNA - 5' end


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_10

description:
matrix column name: Foxa1/2-rep4-D3981
D3981_RNAseq

value: distal colon

value: Mixed background

value: 10.5 months


title: 1097-5 SMAD4- veh 2


title: A2 HTN

description: 10x Genomics (standard methods)

value: Kidney

value: 10-14 weeks

value: angiotensin II-induced HTN


title: A2 Ctrl

description: 10x Genomics (standard methods)

value: angiotensin II-induced control (saline)


title: embryo13_cell7

description: lane1_Ferdinand_Embryos_Plate1_B02_TCGTTAGC

value: Female


title: embryo13_cell6

description: lane1_Ferdinand_Embryos_Plate1_B01_TATGTGGC

value: Blastocyst


title: nanoPARE:Il14H-2mm-rep4

description:
cleavage_sites_miRNA_Il14H_2mm_4.tsv.gz
cleavage_sites_phasiRNA_Il14H_2mm_4.tsv.gz


title: nanoPARE: ago18HM-0.5mm-rep1

description:
cleavage_sites_miRNA_ago18HM_0.5mm_1.tsv.gz
cleavage_sites_phasiRNA_ago18HM_0.5mm_1.tsv.gz


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_4

description:
matrix column name: Control-rep4-D4133
D4133_RNAseq

value: control

value: Male

value: 11 months


title: embryo13_cell10

description: lane1_Ferdinand_Embryos_Plate1_B05_TGACAGAC

value: ICM

value: WT


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_2

description:
matrix column name: Control-rep2-D3853
D3853_RNAseq

value: Foxa1lox/lox; Foxa2lox/lox

value: 9 months


title: nanoPARE: ago18HM-0.5mm-rep2

description:
cleavage_sites_miRNA_ago18HM_0.5mm_2.tsv.gz
cleavage_sites_phasiRNA_ago18HM_0.5mm_2.tsv.gz

value: anther

value: mostly B73

value: polyA RNA - 5' end


title: 1095-5 SMAD4- veh 1

value: Papillomas

value: DMSO


title: 1098-2 SMAD4+ SB 3


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_3

description:
matrix column name: Control-rep3-D3942
D3942_RNAseq

value: distal colon

value: Mixed background

value: 10.5 months


title: 1097-2 SMAD4+ SB 2


title: embryo13_cell5

description: lane1_Ferdinand_Embryos_Plate1_A05_TAGCTTGT

value: Female


title: embryo13_cell4

description: lane1_Ferdinand_Embryos_Plate1_A04_ACTTGATG

value: Blastocyst


title: embryo13_cell3

description: lane1_Ferdinand_Embryos_Plate1_A03_GCCAATGT


title: nanoPARE:Il14H-0.4mm-rep5

description:
cleavage_sites_miRNA_Il14H_0.4mm_5.tsv.gz
cleavage_sites_phasiRNA_Il14H_0.4mm_5.tsv.gz


title: RNA-Seq of mus musculus: adult distal colon of control and FOXA1/2 KO_rep_1

description:
matrix column name: Control-rep1-D3841
D3841_RNAseq

value: control

value: Villin1-Cre; Foxa1lox/wt; Foxa2lox/wt


title: nanoPARE:Il14H-0.4mm-rep4

description:
cleavage_sites_miRNA_Il14H_0.4mm_4.tsv.gz
cleavage_sites_phasiRNA_Il14H_0.4mm_4.tsv.gz


title: 1095-2 SMAD4+ SB 1


title: embryo13_cell8

description: lane1_Ferdinand_Embryos_Plate1_B03_TGTTCTCC


title: RNA-seq_NT_WT_25w_182_Rep 4

city: Illkirch

first: Yvon

last: Trottier

address: 1 rue Laurent Fries

country: France

institute: IGBMC - CNRS UMR 7104 - Inserm U 1258 - University of Strasbourg

data_processing:
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq 2.20.0.422.
According to Illumina Stranded mRNA Prep Ligation - Reference Guide - PN 1000000124518, the first base of each read was trimmed. Reads were preprocessed using cutadapt version 1.10 in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20) and reads shorter than 40 bases were discarded for further analysis. Reads mapping to rRNA were also discarded (this mapping was performed using bowtie version 2.2.8).
Reads were then mapped onto the mm10 assembly of mouse genome using STAR version 2.5.3a.
Gene expression was quantified using htseq-count version 0.6.1p1 and gene annotations from Ensembl release 102.
Assembly: mm10
Supplementary files format and content: tab-delimitated text file includes raw count for each sample

source_name: cerebellum

extract_protocol:
Cerebella were flash frozen in liquid nitrogen and stored at -800C. The total RNA was obtained using TRI Reagent.
RNA-Seq libraries were generated according to manufacturer's instructions from 200 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation.

value: cerebellum


title: nanoPARE:Il14H-2mm-rep3

description:
cleavage_sites_miRNA_Il14H_2mm_3.tsv.gz
cleavage_sites_phasiRNA_Il14H_2mm_3.tsv.gz


title: 1098-1 SMAD4+ veh 3


title: 1095-7-1 SMAD4+ veh 2


title: embryo13_cell2

description: lane1_Ferdinand_Embryos_Plate1_A02_TGACCACT

value: ICM


title: RNA-seq_NT_WT_25w_172_Rep 3

value: WT


title: RNA-seq_NT_WT_25w_171_Rep 2


title: embryo13_cell1

description: lane1_Ferdinand_Embryos_Plate1_A01_CGATGTTT


title: 1095-1 SMAD4+ veh 1


title: nanoPARE:Il14H-0.4mm-rep3

description:
cleavage_sites_miRNA_Il14H_0.4mm_3.tsv.gz
cleavage_sites_phasiRNA_Il14H_0.4mm_3.tsv.gz


title: RNA-seq_NT_WT_25w_170_Rep 1


title: nanoPARE: Oh43-2mm-rep3

description:
cleavage_sites_miRNA_Oh43_2mm_3.tsv.gz
cleavage_sites_phasiRNA_Oh43_2mm_3.tsv.gz


title: RNA_Osr2OE_rep3

city: Xiamen

first: Dawang

last: Zhou

state: Fujian

address: Xiang'an south rd, Xiang'an district

department: Life Science

institute: Xiamen University

data_processing:
Basecalls were performed using CASAVA version 1.8
Sequenced reads were trimmed for adaptor sequence and low quality bases using cutadapt(v.2.6)
Clean reads were mapped to mm10 refernece genome using STAR (v.2.7.5a) .
Read counts were obtained by mapping the aligned reads to the transcripts of known genes from UCSC by using GenomicAlignments (v.1.30.0).
Differential expression analysis was performed with methods based on the negative binomial distribution in DESeq2 (v.1.34.0), using default parameters. Benjamini and Hochberg correction were used for significance tests and multiple comparisons.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw counts and FPKM values for each gene in each sample.

source_name: primary CD8 T cell

growth_protocol: CD8+ T cells were enriched from the spleens and peripheral lymph nodes of donor mice using a naïve CD8+ T cell isolation kit. Cells were resuspended at 106 per ml in RPMI1640 complete medium with 10% FBS (Gibco), 50 M β-mercaptoethanol, 100 U ml-1 penicillin-streptomycin, non-essential amino acids (Gibco) and sodium pyruvate (Gibco).

extract_protocol:
Total RNA was isolated using RaPure Total RNA Micro Kit (Magen, R4012).
The cDNA libraries were constructed from 1 mg RNA sample using mRNA-seq Lib Prep Kit for illumina (ABclonal, RK20302) following the guidelines of the manufacturers.

treatment_protocol: After 24 hours of activation by anti-CD3/CD28, 3 million activated CD8+ T cells were resuspended in the mix of 1 ml fresh 1640 complete RPMI and 3 ml viral supernatant containing 1g ml-1 anti-CD3 and 1 g ml-1 anti-CD28, 50 U ml-1 IL-2 and Polybrene (YEASEN), transferred to 6-well plates, and spun at 800g, 30°C for 60 min. Retroviral supernatants were produced by transfecting the retroviral packaging cell line Plat-Eco (Cell Biolabs) with a retrovirus expression plasmid using Trans-IT (Mirus). This process was repeated the next day. Transduced T cells were cultured for 6 additional days, replacing media and adding fresh IL-2 (50 U ml-1) every day

value: primary CD8 T cell

value: pMIG-Osr2 transduced


title: RNA-seq_shSCR_SCA7_25w_190_Rep 5

value: SCA7 140Q/5Q knock-in

value: shSCR RNA


title: RNA_Osr2OE_rep2


title: embryo12_cell4

value: Male


title: RNA-seq_shSCR_SCA7_25w_173_Rep 3


title: nanoPARE: Oh43-0.4mm-rep2

description:
cleavage_sites_miRNA_Oh43_0.4mm_2.tsv.gz
cleavage_sites_phasiRNA_Oh43_0.4mm_2.tsv.gz

value: anther

value: polyA RNA - 5' end


title: RNA_Osr2OE_rep1


title: embryo12_cell5


title: RNA-seq_shSCR_SCA7_25w_180_Rep 4


title: nanoPARE: Oh43-2mm-rep2

description:
cleavage_sites_miRNA_Oh43_2mm_2.tsv.gz
cleavage_sites_phasiRNA_Oh43_2mm_2.tsv.gz


title: embryo12_cell2


title: embryo12_cell3


title: embryo12_cell1


title: RNA_Vector_rep2

value: pMIG-Vector transduced


title: RNA-seq_shSCR_SCA7_25w_169_Rep 2


title: nanoPARE: NC358-2mm-rep3

description:
cleavage_sites_miRNA_NC358_2mm_3.tsv.gz
cleavage_sites_phasiRNA_NC358_2mm_3.tsv.gz


title: RNA-seq_shA4_SCA7_25w_181_Rep 5

value: shA4 RNA


title: nanoPARE: NC358-2mm-rep2

description:
cleavage_sites_miRNA_NC358_2mm_2.tsv.gz
cleavage_sites_phasiRNA_NC358_2mm_2.tsv.gz


title: nanoPARE: Oh43-2mm-rep1

description:
cleavage_sites_miRNA_Oh43_2mm_1.tsv.gz
cleavage_sites_phasiRNA_Oh43_2mm_1.tsv.gz


title: embryo11_cell4


title: embryo11_cell3


title: RNA-seq_shA4_SCA7_25w_167_Rep 4


title: nanoPARE: NC358-2mm-rep1

description:
cleavage_sites_miRNA_NC358_2mm_1.tsv.gz
cleavage_sites_phasiRNA_NC358_2mm_1.tsv.gz


title: RNA_Vector_rep1


title: ATAC_Osr2OE_rep2

data_processing:
Raw ATAC-seq reads were trimmed and filtered for quality using CutAdapt (v.2.6). Paired-end reads were aligned using Bowtie2 (v.2.3.5.1) (PMID: 22388286) against mm10 genome.
Aligned reads were sorted and non-uniquely mapping reads were marked using Picard (v.2.9.5) (http://broadinstitute.github.io/picard/). PCR duplicates, reads mapped to mitochondria or of low mapping quality were filtered using Samtools (v.1.9) (PMID: 19505943).
To correct for the fact that the Tn5 transposase binds as a dimer and inserts two adapters in the Tn5 tagmentation step, all positive-strand reads were shifted 4 bp downstream and all negative-strand reads were shifted 5 bp upstream to center the reads on the transposon binding event.
We then pooled the shifted reads by sample type and identified peaks using MACS2 (v.2.2.6) (PMID: 18798982) with a threshold of FDR-corrected p < 0.001 using the Benjamini-Hochberg procedure for multiple hypothesis correction. A union peak list for each experiment was created by combining all peaks in all samples; overlapping peaks were merged with minimum overlap > 0.5. The number of reads in each peak was determined with Diffbind (v.3.6.3) (PMID: 22217937).
Differentially expressed peaks were identified after DESeq2 normalization using a FDR cut-off of 0.05. Peaks were annotated using R package ChIPseeker (v.1.18.0) (PMID: 25765347).
Assembly: mm10
Supplementary files format and content: BigWig Track Format of each ATAC-seq sample.

extract_protocol:
Nuclei from 3 × 10^5 cells were isolated using a lysis buffer composed of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin. Immediately after cell lysis, nuclei were pelleted in low-bind 1.5-ml tubes (Eppendorf) and resuspended in transpose mix (Vazyme)
Transposition was performed at 37 °C for 30 min. After purification of the DNA with the VAHTS DNA Clean Beads (Vazyme) at a 2 × ratio, libraries were barcoded using TruePrep™ Index Kit V2 for lllumina (Vazyme TD202) and amplified for 5 cycles. Additional PCR cycles were evaluated by real time PCR. Final product was cleaned and size selected by VAHTS DNA Clean Beads at a 0.6 × / 0.7 × ratio.


title: RNA-seq_shA4_SCA7_25w_166_Rep 3


title: nanoPARE: NC358-0.4mm-rep2

description:
cleavage_sites_miRNA_NC358_0.4mm_2.tsv.gz
cleavage_sites_phasiRNA_NC358_0.4mm_2.tsv.gz


title: ATAC_Vector_rep2


title: RNA_Vector_rep3


title: RNA-seq_shSCR_SCA7_25w_168_Rep 1


title: RNA-seq_shA4_SCA7_25w_163_Rep 1


title: nanoPARE: Oh43-0.4mm-rep1

description:
cleavage_sites_miRNA_Oh43_0.4mm_1.tsv.gz
cleavage_sites_phasiRNA_Oh43_0.4mm_1.tsv.gz


title: RNA-seq_shA4_SCA7_25w_164_Rep 2


title: ATAC_Vector_rep1


title: nanoPARE: HP301-2mm-rep2

description:
cleavage_sites_miRNA_HP301_2mm_2.tsv.gz
cleavage_sites_phasiRNA_HP301_2mm_2.tsv.gz


title: embryo11_cell2


title: nanoPARE: NC358-0.4mm-rep1

description:
cleavage_sites_miRNA_NC358_0.4mm_1.tsv.gz
cleavage_sites_phasiRNA_NC358_0.4mm_1.tsv.gz


title: RNA_WT_Yoda1_rep3

treatment_protocol: After 24 hours of activation by anti-CD3/CD28, activated CD8+ T cells were treatment with DMSO or Yoda1 (10 M) for another 24h.


title: embryo10_cell3

description: lane1_Ferdinand_Embryos_Plate2_H01_ACTGGTCG


title: nanoPARE: HP301-2mm-rep1

description:
cleavage_sites_miRNA_HP301_2mm_1.tsv.gz
cleavage_sites_phasiRNA_HP301_2mm_1.tsv.gz


title: RNA-seq_WT_190_9w_NT_Rep 3


title: embryo11_cell1


title: embryo11_cell5


title: RNA-seq_WT_181_9w_NT_Rep 2


title: embryo10_cell2

description: lane1_Ferdinand_Embryos_Plate2_G01_ACTTTACT


title: RNA_WT_Yoda1_rep2


title: nanoPARE: HP301-0.4mm-rep2

description:
cleavage_sites_miRNA_HP301_0.4mm_2.tsv.gz
cleavage_sites_phasiRNA_HP301_0.4mm_2.tsv.gz


title: embryo10_cell1

description: lane1_Ferdinand_Embryos_Plate2_A02_TAGTGACT


title: RNA_WT_Yoda1_rep1


title: RNA_WT_DMSO_rep3

value: DMSO


title: RNA-seq_WT_179_9w_NT_Rep 1


title: nanoPARE: HP301-0.4mm-rep1

description:
cleavage_sites_miRNA_HP301_0.4mm_1.tsv.gz
cleavage_sites_phasiRNA_HP301_0.4mm_1.tsv.gz


title: RNA-seq_WT_173_9w_shA4_Rep 3


title: embryo9_cell6

description: lane1_Ferdinand_Embryos_Plate2_F01_GTCTGAGG

value: Female


title: ATAC_Osr2OE_rep1


title: nanoPARE: B73-2mm-rep2

description:
cleavage_sites_miRNA_B73_2mm_2.tsv.gz
cleavage_sites_phasiRNA_B73_2mm_2.tsv.gz


title: RNA_WT_DMSO_rep2


title: RNA-seq_WT_8_9w_shA4_Rep 2


title: embryo9_cell3

description: lane1_Ferdinand_Embryos_Plate2_C01_CCTAGTAT

value: Blastocyst


title: nanoPARE: B73-2mm-rep1

description:
cleavage_sites_miRNA_B73_2mm_1.tsv.gz
cleavage_sites_phasiRNA_B73_2mm_1.tsv.gz


title: RNA_WT_DMSO_rep1


title: nanoPARE: B73-0.4mm-rep2

description:
cleavage_sites_miRNA_B73_0.4mm_2.tsv.gz
cleavage_sites_phasiRNA_B73_0.4mm_2.tsv.gz


title: embryo9_cell4

description: lane1_Ferdinand_Embryos_Plate2_D01_CGGAAACA


title: RNA-seq_WT_1_9w_shA4_Rep 1


title: PBMCs_TAC-treated_mouse_rep4

data_processing:
The NextSeq RTA (version 2.4.11.0) was used for base calling. bcl2fastq (version 2.20.0) was used to convert the .bcl files into .fastq files. Fastq files were aligned to the mouse reference genome (mm10) using HISAT2 software and assembled using StringTie. R package DESeq2 was used for data normalization and differential expression analysis.
Assembly: mm10
Supplementary files format and content: raw counts

source_name: Mouse PBMCs from TAC-treated mouse

treatment_protocol: Mouse was treated with DMSO or TAC.

value: Mouse PBMCs

value: TAC-treated


title: embryo9_cell2

description: lane1_Ferdinand_Embryos_Plate2_B01_GCAACATT


title: scRNA_OT-I_rep1

data_processing:
Basecalls were performed using CASAVA version 1.8
All following steps were performed using the CellRanger analysis pipeline (v.7.0.1).
Sample demultiplexing, barcode processing
alignment, filtering
UMI counting and aggregation of sequencing runs
Assembly: mm10
Supplementary files format and content: Raw features, barcodes, and matrices (raw_feature_bc_matrix) for each library.

growth_protocol: CD8+ T cells were enriched from the spleens and peripheral lymph nodes of donor mice using a naïve CD8+ T cell isolation kit.

extract_protocol:
Two weeks after adoptive transfer, WT (OT-I CD45.1-CD45.2+) T cells were flow-sorted. OT-I T cells from two mice were pooled for one library preparation.
Cells were counted and loaded onto the Chromium Controller (10 × Genomics) for a target recovery of 1 × 10^4 single cells. Samples were processed per the manufacturer’s protocol and sequenced on an Illumina NovaSeq6000 sequencer using a 150-bp kit with paired-end reads.

treatment_protocol: For subcutaneous tumor models, MC38-OVA (1 × 10^6) tumor cells were injected subcutaneously into the flanks of WT recipient mice (CD45.1+). Tumor-engrafted recipient mice were first intraperitoneally injected with 200 mg kg-1 cyclophosphamide (CTX, Aladdin) when the tumor is palpable, then OT-I naive T (1x10^6) cells were adoptively transferred by tail-vein injection into recipient mice.

value: MC38-OVA bearing


title: embryo9_cell1

description: lane1_Ferdinand_Embryos_Plate2_A01_TGCGATCT

value: ICM


title: scRNA_OT-I_rep2


title: Smart-seq2: ago18HT-5mm-rep3

value: mostly B73

value: polyA RNA - transcript body


title: nanoPARE: B73-0.4mm-rep1

description:
cleavage_sites_miRNA_B73_0.4mm_1.tsv.gz
cleavage_sites_phasiRNA_B73_0.4mm_1.tsv.gz


title: PBMCs_TAC-treated_mouse_rep2

value: WT


title: PBMCs_TAC-treated_mouse_rep3


title: RNA_Osr2-KO_rep2

extract_protocol:
Two weeks after adoptive transfer, WT (Osr2fl/fl OT-I CD45.1-CD45.2+) and Osr2 knockout (Osr2fl/fl Lck-Cre OT-I CD45.1+CD45.2+) T cells were flow-sorted. Total RNA was isolated using RaPure Total RNA Micro Kit (Magen, R4012).
The cDNA libraries were constructed from 10-100 ng RNA sample using Whole RNA-seq Lib Prep kit for illumina (ABclonal, RK20303) following the guidelines of the manufacturers.

treatment_protocol: For subcutaneous tumor models, MC38-OVA (1 × 10^6) tumor cells were injected subcutaneously into the flanks of WT recipient mice (CD45.1+). Tumor-engrafted recipient mice were first intraperitoneally injected with 200 mg kg-1 cyclophosphamide (CTX, Aladdin) when the tumor is palpable, then Osr2fl/fl OT-I T cells were mixed 1:1 with congenically disparate OT-I T cells of the desired genotype (Osr2fl/fl Lck-Cre OT-I) and a total of 1×10^6 naïve T cells were adoptively transferred by tail-vein injection into recipient mice.

value: primary CD8 T cell

value: Osr2fl/fl Lck-Cre OT-I CD45.1+CD45.2+


title: PBMCs_TAC-treated_mouse_rep1


title: Smart-seq2: ago18HT-5mm-rep2


title: embryo8_cell10


title: embryo8_cell9


title: PBMCs_DMSO-treated_mouse_rep4

source_name: Mouse PBMCs from DMSO-treated mouse

value: DMSO-treated


title: RNA_Osr2-KO_rep1


title: Smart-seq2: ago18HT-5mm-rep1

value: anther


title: Smart-seq2: ago18HT-2mm-rep3


title: RNA_WT_rep3

value: Osr2fl/fl OT-I CD45.1-CD45.2+


title: embryo9_cell5

description: lane1_Ferdinand_Embryos_Plate2_E01_CGCACGAG


title: embryo8_cell8


title: PBMCs_DMSO-treated_mouse_rep3


title: Smart-seq2: ago18HT-2mm-rep2


title: embryo8_cell7


title: RNA_WT_rep2


title: embryo8_cell6


title: PBMCs_DMSO-treated_mouse_rep2


title: RNA_WT_rep1


title: Smart-seq2: ago18HT-2mm-rep1


title: PBMCs_DMSO-treated_mouse_rep1


title: Smart-seq2: ago18HT-0.5mm-rep2


title: embryo8_cell5


title: RNA_Osr2-KO_rep3


title: ATAC_Piezo1-KO_Yoda1_rep2


title: embryo8_cell4


title: zur 3 600ug/mL s1 calprotectin

first: Eric

middle: P

last: Skaar

address: 1161 21st Avenue South

department: Pathology, Microbiology, and Immunology

data_processing:
Bioinformatic analysis was performed using CLC Genomics Workbench Software. Paired-end sequencing data in fastq.gz format were imported into CLC Genomics Workbench and trimmed to remove barcodes and adapter sequences. The remaining ribosomal RNA reads were depleted computationally by aligning all reads to a sequence list containing all rRNA sequences for the Staphylococcus aureus genome (NC_009641). Unmapped (non-rRNA) reads were collected and utilized for “RNA-seq analysis” function (standard settings, similarity fraction of 0.8) to generate expression values for each gene.
Assembly: Staphylococcus aureus genome (NC_009641)
Supplementary files format and content: Excel file with differential expression against vehicle for all the strains. Included Log2 fold change and P-value

organism: Staphylococcus aureus

source_name: Bacteria

extract_protocol:
QIAGEN RNeasy
NEBNext Ultra II RNA Library Prep kit for Illumina and NEBNext Multiplex Oligos for Illumina

value: Newman

value: Bacteria

value: zur

value: 600ug/mL s1 calprotectin


title: zur 1 600ug/mL s1 calprotectin


title: Smart-seq2: ago18HT-0.5mm-rep1


title: zur 2 600ug/mL s1 calprotectin


title: ATAC_Piezo1-KO_Yoda1_rep3


title: embryo8_cell3

data_processing:
Raw reads were processed as previously (https://doi.org/10.1038/s41467-018-03149-4), aligning to the GRCh38 reference genome using Hisat2 (https://doi.org/10.1038/s41587-019-0201-4).
Gene expression counts were computed from the mapped data using the featureCounts function within RSubread (https://doi.org/10.1093/nar/gkz114).
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text file with raw counts for each sample

value: Blastocyst

value: Female


title: mntR 3 600ug/mL s1 calprotectin

value: mntR


title: ATAC_Piezo1-KO_DMSO_rep3

value: DMSO


title: embryo8_cell2

value: ICM


title: Smart-seq2: ago18HM-5mm-rep2

value: mostly B73


title: Smart-seq2: ago18HM-5mm-rep1

value: polyA RNA - transcript body


title: embryo8_cell1

value: WT


title: ATAC_Piezo1-KO_DMSO_rep2

value: primary CD8 T cell


title: mntR 2 600ug/mL s1 calprotectin


title: Smart-seq2: ago18HM-2mm-rep3


title: embryo7_cell9

description: lane1_Ferdinand_Embryos_Plate2_H04_GCTTAAAC

value: Male


title: ATAC_WT_Yoda1_rep3


title: mntR 1 600ug/mL s1 calprotectin


title: Smart-seq2: ago18HM-2mm-rep2


title: ATAC_Piezo1-KO_DMSO_rep1


title: embryo7_cell8

description: lane1_Ferdinand_Embryos_Plate2_G04_GCGTGAAC


title: embryo7_cell7

description: lane1_Ferdinand_Embryos_Plate2_F04_GATCGAAC


title: Smart-seq2: ago18HM-5mm-rep3


title: fur 2 600ug/mL s1 calprotectin

value: fur


title: fur 3 600ug/mL s1 calprotectin


title: Smart-seq2: ago18HM-2mm-rep1

value: anther


title: ATAC_WT_Yoda1_rep2


title: ATAC_Piezo1-KO_Yoda1_rep1


title: fur 1 600ug/mL s1 calprotectin


title: embryo7_cell5

description: lane1_Ferdinand_Embryos_Plate2_D04_CTCAGATA


title: ATAC_WT_Yoda1_rep1


title: embryo7_cell6

description: lane1_Ferdinand_Embryos_Plate2_E04_AGGTCTAA


title: Smart-seq2: ago18HM-0.5mm-rep3


title: ATAC_WT_DMSO_rep3


title: WT 3 600ug/mL s1 calprotectin


title: embryo7_cell4

description: lane1_Ferdinand_Embryos_Plate2_C05_CACTCGAG


title: Smart-seq2: ago18HM-0.5mm-rep1


title: ATAC_WT_DMSO_rep2


title: embryo7_cell3

description: lane1_Ferdinand_Embryos_Plate2_C04_CTGTACGG


title: ATAC_WT_DMSO_rep1


title: WT 1 600ug/mL s1 calprotectin


title: Smart-seq2:Il14H-2mm-rep3


title: embryo7_cell2

description: lane1_Ferdinand_Embryos_Plate2_B05_GGAATGAT


title: zur 3 300ug/mL WT calprotectin

value: 300ug/mL WT calprotectin


title: scRNA-seq_αLP_hashtag

city: Chiba-shi

first: Arifumi

last: Iwata

state: Chiba

address: 1-8-1 Inohana, Chuou-ku

department: Department of Allergy and Clinical Immunology

country: Japan

institute: Chiba University

library_source: other

data_processing:
After adaptor removal by Cutadpat, gene-expression libraries were aligned to the mm10 reference transcriptome by Bowtie2, and count matrices were generated using modified BD Rhapsody workflow. Valid cell barcodes were identified as cell barcodes above the inflection threshold of the knee-plot of total read counts of each cell barcode identified by the DropletUtils package. Sample origins and doublets were identified based on fold change of the normalized read counts of the hashtags. The count matrices were imported and analyzed using Seurat v.4.
Assembly: GRCm38
Supplementary files format and content: count matrix

molecule: protein

source_name: Bone Marrow

extract_protocol:
Bone marrow cells were harvested from the cut-off of both ends of the femur, tibia and pelvis, humerus, and scapula of mice using a centrifuge. Lineage-positive cells were depleted using a MOJO Hematopoietic Isolation Kit. αLP were sorted using FACSAriaII.
Hashtag labeled sorted cells were combined, trapped, and reverse-transcribed using BD Rhapsody (BD) according to the manufacturer's instructions. Single-cell RNA sequencing was performed using the platform of TAS-Seq.

value: WT and G3SEKO

value: Bone Marrow

value: lymphoid progenitor

tag: antibodies/tags

value: Anti-MHC classI,  WT:TotalSeq-A307, A308, A309; G3SEKO:TotalSeq-A310, A311, A312)


title: embryo7_cell1

description: lane1_Ferdinand_Embryos_Plate2_A05_TCGAAGTG

value: Blastocyst


title: Smart-seq2:Il14H-0.4mm-rep4


title: embryo6_cell10

description: lane1_Ferdinand_Embryos_Plate2_H03_CGTATGCA


title: scRNA-seq_αLP_mRNA


title: Smart-seq2: ago18HM-0.5mm-rep2


title: Smart-seq2:Il14H-0.4mm-rep5


title: zur 1 300ug/mL WT calprotectin

value: Newman

value: Bacteria

value: zur


title: 8 week mouse IVD [SM22-8W-1-4_S4]

city: Pok Fu Lam

first: Peikai

last: Chen

address: Sassoon Road 5

department: Faculty of Medicine

country: Hong Kong

institute: The University of Hong Kong

description: 10X single-cell RNA-seq

data_processing:
Data was aligned to mouse genome mm10 (reference v3.0.0 provided by 10X Genomics) by CellRanger (version 3.1.0).
Data was also aligned to a reference file of exogenous sequences, including tdTomato (1431bp), by CellRanger (version 3.1.0).
The resulting matrix files were integrated and further processed on Seurat (version 3).
Dimension reduction including CCA, PCA, tSNE and UMAP were performed.
Clustering were performed, and differentially expressed genes between one cluster and another (or all remaining clusters) were performed. A further filtering of abs(log2(fold-change)) > 1/2 and FDR <0.05 was applied to ensure higher specificity and correct for multiple testing.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: gzip text files of gene expression.
Supplementary_files_format_and_content: P10_barcodes.tsv.gz: Gene expression file in mm10, for the p10 sample.
Supplementary_files_format_and_content: P10_features.tsv.gz: Gene expression file in mm10, for the p10 sample.
Supplementary_files_format_and_content: P10_matrix.mtx.gz: Gene expression file in mm10, for the p10 sample.
Supplementary_files_format_and_content: P10_exo_barcodes.tsv.gz: Gene expression file in exogenous genes, for the p10 sample.
Supplementary_files_format_and_content: P10_exo_features.tsv.gz: Gene expression file in exogenous genes, for the p10 sample.
Supplementary_files_format_and_content: P10_exo_matrix.mtx.gz: Gene expression file in exogenous genes, for the p10 sample.
Supplementary_files_format_and_content: 8W_barcodes.tsv.gz: Gene expression file in mm10, for the 8WK sample.
Supplementary_files_format_and_content: 8W_features.tsv.gz: Gene expression file in mm10, for the 8WK sample.
Supplementary_files_format_and_content: 8W_matrix.mtx.gz: Gene expression file in mm10, for the 8WK sample.
Supplementary_files_format_and_content: 8W_exo_barcodes.tsv.gz: Gene expression file in exogenous genes, for the 8WK sample.
Supplementary_files_format_and_content: 8W_exo_features.tsv.gz: Gene expression file in exogenous genes, for the 8WK sample.
Supplementary_files_format_and_content: 8W_exo_matrix.mtx.gz: Gene expression file in exogenous genes, for the 8WK sample.

source_name: whole IVD at the lumbar and the tail

growth_protocol: The mice grew under normal condition.

extract_protocol:
RNA extraction from disassociated cells was done according to standard 10X Genomics Protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: No special treatment was applied.

value: whole IVD


title: mntR 3 300ug/mL WT calprotectin

value: mntR


title: Smart-seq2: Oh43-2mm-rep3

value: polyA RNA - transcript body


title: Smart-seq2:Il14H-0.4mm-rep3

description: cleavage_sites_phasiRNA_Il14H_0.4mm_3.tsv.gz


title: WT 2 600ug/mL s1 calprotectin

value: WT

value: 600ug/mL s1 calprotectin


title: embryo6_cell9

description: lane1_Ferdinand_Embryos_Plate2_G03_TCACCATT

value: ICM

value: Male


title: Smart-seq2: Oh43-2mm-rep2

value: anther


title: 8 week mouse IVD [SM22-8W-1-1_S1]

description: 10X single-cell RNA-seq


title: mntR 1 300ug/mL WT calprotectin

value: 300ug/mL WT calprotectin


title: Smart-seq2:Il14H-2mm-rep4


title: mntR 2 300ug/mL WT calprotectin


title: embryo6_cell7

description: lane1_Ferdinand_Embryos_Plate2_E03_AGGACGAT


title: p10 mouse IVD [SM22a-P10-1-4_S84]

description: 10X single-cell RNA-seq


title: embryo6_cell6

description: lane1_Ferdinand_Embryos_Plate2_D03_CATGGGAA


title: fur 3 300ug/mL WT calprotectin

value: fur


title: Smart-seq2: Oh43-0.4mm-rep2


title: zur 2 300ug/mL WT calprotectin


title: embryo6_cell5

description: lane1_Ferdinand_Embryos_Plate2_C03_CATGAATG


title: p10 mouse IVD [SM22a-P10-1-3_S83]

description: 10X single-cell RNA-seq


title: fur 2 300ug/mL WT calprotectin


title: embryo6_cell4

description: lane1_Ferdinand_Embryos_Plate2_B04_GTTAGCCT


title: embryo6_cell3

description: lane1_Ferdinand_Embryos_Plate2_B03_GAGGTGCT


title: fur 1 300ug/mL WT calprotectin


title: p10 mouse IVD [SM22a-P10-1-2_S82]

description: 10X single-cell RNA-seq


title: 8 week mouse IVD [SM22-8W-1-3_S3]

description: 10X single-cell RNA-seq


title: embryo6_cell8

description: lane1_Ferdinand_Embryos_Plate2_F03_AGTTGAAA


title: 8 week mouse IVD [SM22-8W-1-2_S2]

description: 10X single-cell RNA-seq


title: Smart-seq2: Oh43-0.4mm-rep1


title: embryo6_cell2

description: lane1_Ferdinand_Embryos_Plate2_A04_TAGTCTTG


title: p10 mouse IVD [SM22a-P10-1-1_S81]

description: 10X single-cell RNA-seq


title: WT 2 300ug/mL WT calprotectin


title: Animal 3, section 1, MERFISH

type: other

city: Munich

first: Peter

last: Androvic

state: Bavaria

address: Feodor-Lynen-Strasse 17

country: Germany

institute: LMU Hospital, Institute for Stroke and Dementia Research

description:
panel of 496 genes was measured by MERFISH
MERFISH_5xFAD_expression.txt
MERFISH_5xFAD_metadata.txt

data_processing:
Brain sections were measured by MERFISH according to the manufacturer's protocol (https://vizgen.com/technology/#merfish)
Encoded MERFISH images were processed according to manufacturer's pipeline (https://vizgen.com/technology/#merfish). The output of this pipeline are spatial positions of individual transcripts and segmented single cells based on DAPI and poly-A stainings. Single cell expression tables are produced by counting individual transcripts within segmented cell boundaries.

molecule: other

source_name: brain cells

extract_protocol:
Brain sections, 10 μm in thickness, were cut coronally using a cryotome (CryoStar NX70, Thermo Scientific) and collected onto glass slides supplied by Vizgen Corp. (Cambridge, MA 02138; MERSCOPE slide part number 20400001). To ensure proper adhesion, the glass slides were coated with a solution of 0.1 mg/mL poly-D-lysine bromide (Sigma, P7886) and left to incubate at room temperature for 1 hour and dried before starting the sectioning.
MERFISH sample preparation was performed according to manufacturer's protocol (https://vizgen.com/resources/fresh-and-fixed-frozen-tissue-sample-preparation)

treatment_protocol: Male 5xFAD and litter-mate mice at 9.5 months old were anesthetized using an Isoflurane chamber and filmcontainer filled with Isoflurane-soaked cotton fibers and perfused with RNase-free PBS (Sigma, D8537). Next, the brains were carefully extracted, cut in half and and frozen in isopentane on dry ice. The brains were then stored at −80 °C until further processing.

value: Brain

value: C57BL/6J, 5xFAD

value: 9.5 months


title: Smart-seq2: NC358-2mm-rep1


title: Animal 2, section 2, MERFISH


title: Smart-seq2: Oh43-2mm-rep1


title: WT 1 300ug/mL WT calprotectin


title: Smart-seq2: NC358-0.4mm-rep2


title: embryo5_cell7

description: lane1_Ferdinand_Embryos_Plate2_H02_AGCGATGA

extract_protocol:
Cells were collected directly into 2.5 μl methyltransferase reaction mixture which was comprised of 1 × M.CviPI Reaction buffer, 2 U M.CviPI, 160 μM S-adenosylmethionine, 1 U μl−1 RNAsein, 0.1% IGEPAL then incubated for 15 min at 37 °C. The reaction was stopped and the RNA preserved with the addition of 5 μl RLT plus.
Single-cell RNA sequencing libraries were prepared according to the RNA half of the scNMT-seq protocol (https://doi.org/10.1038/s41467-018-03149-4). Briefly, biotinylated oligo-dT was bound to magnetic beads which were added to single-cell lysates in 96w plates. The captured mRNA was then reverse transcribed and PCR amplified on beads accordingto the Smartseq2 protocol (https://doi.org/10.1038/nprot.2016.138,https://doi.org/10.1038/nprot.2014.006). Indexed Illumina libraries were prepared from the amplified cDNA using Nextera XT reagents and then pooled and sequenced on a HiSeq 2000 instrument using 75bp paired-end reads.

value: Blastocyst


title: Animal 2, section 1, MERFISH


title: zur 3 No calprotectin

value: Newman

value: Vehicle


title: Smart-seq2: NC358-0.4mm-rep1


title: embryo5_cell6

description: lane1_Ferdinand_Embryos_Plate2_G02_ATAACTAG


title: Animal 1, section 2, MERFISH


title: zur 2 No calprotectin

value: Bacteria

value: zur


title: Smart-seq2: NC358-2mm-rep3


title: embryo5_cell5

description: lane1_Ferdinand_Embryos_Plate2_F02_AATTTGCG


title: zur 1 No calprotectin


title: Smart-seq2: NC358-2mm-rep2


title: Animal 1, section 1, MERFISH


title: WT 3 300ug/mL WT calprotectin


title: Smart-seq2: HP301-2mm-rep1


title: Drosophila testes, 72h PHS, Polysome fraction '2-3 ribosomes', Replicate 2

city: Stanford

first: Cameron

middle: W

last: Berry

state: California

address: 279 W. Campus Drive, B300

department: Developmental Biology

institute: Stanford University

description: biological replicate 2

data_processing:
Sequence reads were mapped to dm6 using BOWTIE version 0.12.8 with a value of e = 5000 to allow for expected mismatches in the poly-A tail not encoded in the genome
Custom perl script written to identify sequencing reads that contain polyA sequences that were not genomically encoded
Reads within annotated 3'UTRs (with 500bp extension) were assigned to the nearest upstream CDS end as defined by Flybase
Assembly: dm6
Supplementary files format and content: tab-delimited text files containing in column: 1) location of CDS end, 2) gene symbol, 3) associated cleavage site location and count (location and count separated by *, and multiple cleavage sites are separate by |)

organism: Drosophila melanogaster

source_name: Drosophila testes

growth_protocol: Progeny of fly crosses were grown at 22C in bottles containing standard molasses media

extract_protocol:
For Polysome profiling, RNA was isolated by acid phenol-chloroform extraction and fractions were combined based on the absorbance profiles at A260 nm to create 6 groups: free, 40S, 60S, 80S, 2-3 ribosomes and 4+ ribosomes
RNA libraries for RNA-seq were prepared using QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) following manufacturer's protocols.

treatment_protocol: Pupae were heat shocked by placing bottles in 37C water bath for 30 mins and returned to 25C incubator

value: Drosophila testes

value: bam-/-;hsBam

value: 30 min 37C heat shock

tag: time

value: 72 hours


title: Drosophila testes, 72h PHS, Polysome fraction '4+ ribosomes', Replicate 2

description: biological replicate 2


title: mntR 3 No calprotectin

value: mntR


title: embryo5_cell4

description: lane1_Ferdinand_Embryos_Plate2_E02_AGCGCCAA

value: ICM

value: WT

value: Male


title: embryo6_cell1

description: lane1_Ferdinand_Embryos_Plate2_A03_TCCTCAAT


title: Smart-seq2: HP301-0.4mm-rep1

source_name: anther

value: anther

value: polyA RNA - transcript body


title: mntR 1 No calprotectin


title: Smart-seq2: HP301-0.4mm-rep2


title: Drosophila testes, 72h PHS, Polysome fraction '80S', Replicate 2

description: biological replicate 2


title: mntR 2 No calprotectin


title: embryo5_cell3

description: lane1_Ferdinand_Embryos_Plate2_D02_TAGGACCC

source_name: Blastocyst


title: fur 3 No calprotectin

value: fur


title: Drosophila testes, 72h PHS, Polysome fraction '60S', Replicate 2

description: biological replicate 2


title: embryo5_cell1

description: lane1_Ferdinand_Embryos_Plate2_B02_GAATCTGT


title: embryo5_cell2

description: lane1_Ferdinand_Embryos_Plate2_C02_CTTCACAT


title: Smart-seq2: B73-2mm-rep1


title: Smart-seq2: B73-2mm-rep2


title: embryo4_cell16

description: lane1_Ferdinand_Embryos_Plate2_H07_TCCTCTCT


title: fur 2 No calprotectin


title: Drosophila testes, 72h PHS, Polysome fraction '40S', Replicate 2

description: biological replicate 2


title: Smart-seq2: B73-0.4mm-rep2


title: embryo4_cell15

description: lane1_Ferdinand_Embryos_Plate2_H06_GTACGGAC


title: Smart-seq2: HP301-2mm-rep2


title: Drosophila testes, 72h PHS, Polysome fraction 'free', Replicate 2

description: biological replicate 2


title: fur 1 No calprotectin


title: embryo4_cell14

description: lane1_Ferdinand_Embryos_Plate2_G07_AACAGCGC


title: Smart-seq2: B73-0.4mm-rep1


title: small RNA-seq: ago18HT-5mm-rep3

data_processing:
To quantify phasiRNA abundance, sRNA-seq reads were mapped to the respective Zea genomes using Bowtie v1.3.1; to quantify miRNA abundance, sRNA-seq reads were mapped to a FASTA file of all mature miRNAs annotated for each genome using bowtie; and to quantify mRNA abundance, Smart-seq2 reads were mapped to the respective Zea genomes (B73 RefGen_v5 was used for the ago18 mutant data) using HISAT v2.1.0. Reads mapped to PHAS loci or miRNAs were counted using featureCounts v1.6.3 and normalized to counts per million (CPM) using edgeR v4.0.2. Differential expression analyses were performed using edgeR with a generalized linear model-based method. Small RNA-transcript interactions in each anther sample were identified using the nanoPARE analysis pipeline (Schon et al., 2018). The sRNA-target pairs with adjusted P value < 0.05 at the EndCut step and are detected in at least two biological replicates were considered positive.
Assembly: Respective reference genomes of Zea varieties
Supplementary files format and content: CPM tables for sRNA-seq and RNA-seq, cleavage-site tables for nanoPARE

extract_protocol:
Fresh anthers were snap-frozen in liquid nitrogen and total RNA was extracted using the TRI reagent (Sigma-Aldrich) or the TraPR Small RNA Isolation kit (Lexogen; for TraPR sRNA-seq only)
sRNA-seq libraries were prepared from ~100 ng total RNA per sample using a Somagenics RealSeq-AC miRNA library kit following the manufacturer’s protocol. RNA-seq (Smart-seq2) and nanoPARE libraries were prepared from 5 ng total RNA per sample using the nanoPARE library preparation protocol.

value: mostly B73

value: small RNA - total


title: embryo4_cell13

description: lane1_Ferdinand_Embryos_Plate2_G06_TGGCAAAA

value: Blastocyst


title: Drosophila testes, 48h PHS, Polysome fraction '80S', Replicate 2

description: biological replicate 2

value: 48 hours


title: WT 2 No calprotectin

value: Newman

value: Bacteria

value: Vehicle


title: small RNA-seq: ago18HT-5mm-rep2


title: embryo4_cell12

description: lane1_Ferdinand_Embryos_Plate2_F07_ACTCAGGA


title: embryo4_cell11

description: lane1_Ferdinand_Embryos_Plate2_F06_ATACAGGC


title: Drosophila testes, 48h PHS, Polysome fraction '2-3 ribosomes', Replicate 2

description: biological replicate 2

value: Drosophila testes

value: bam-/-;hsBam


title: small RNA-seq: ago18HT-2mm-rep3


title: embryo4_cell10

description: lane1_Ferdinand_Embryos_Plate2_E07_GTTTTCAC


title: embryo4_cell9

description: lane1_Ferdinand_Embryos_Plate2_E06_TGCTGAAC


title: WT 3 No calprotectin


title: small RNA-seq: ago18HT-2mm-rep2


title: small RNA-seq: ago18HT-5mm-rep1


title: 35SLZ_90FR_rep3

city: Umeå

first: Stephan

last: Wenkel

address: Parallellvägen 21

country: Sweden

institute: Umeå Plant Science Center

data_processing:
BASH command line, FastQC and R
Sequence reads were trimmed for adaptor sequence/low quality sequence using Trimmomatic (Leading = 25, Trailing = 25, MINLEN = 35, Sliding Window = 5:35). Reads quality was checked using FastQC.
Mapping was done using STAR in R
Read counts extraction and normalization were performed in R
The counts from each sample were merged together in a merged count file of that biological replicate, after checking the replicates by PCA
Assembly: TAIR10
Supplementary files format and content: Tab delimited text files combining raw counts of each replicate in one text file per biological sample

organism: Arabidopsis thaliana

source_name: Whole 12 days old seedlings

extract_protocol:
Total RNA was extracted using either the Spectrum Plant Total RNA Kit (Sigma)
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

value: Whole 12 days old seedlings

value: 35::ATHB2:LZ in Col-0 background

value: Homozygous genomic insertion and overexpression of exons 3 and 4 of ATHB2, controlled by a 35S promoter

value: 90 minutes FR enriched light


title: 35SLZ_90FR_rep2


title: Drosophila testes, 48h PHS, Polysome fraction 'free', Replicate 2

description: biological replicate 2

value: 30 min 37C heat shock


title: 35SLZ_90FR_rep1


title: Drosophila testes, 48h PHS, Polysome fraction '40S', Replicate 2

description: biological replicate 2


title: WT 1 No calprotectin

value: WT


title: embryo4_cell8

description: lane1_Ferdinand_Embryos_Plate2_D07_CAAAACCG

value: ICM

value: Male


title: Drosophila testes, 24h PHS, Polysome fraction '4+ ribosomes', Replicate 2

description: biological replicate 2

value: 24 hours


title: small RNA-seq: ago18HT-0.5mm-rep3

value: anther

value: mostly B73

value: small RNA - total


title: embryo4_cell7

description: lane1_Ferdinand_Embryos_Plate2_D06_ACCTTCAA

value: Blastocyst


title: 35S_LZ_45FR_rep3

value: 45 minutes FR-enriched light


title: Drosophila testes, 24h PHS, Polysome fraction '2-3 ribosomes', Replicate 2

description: biological replicate 2


title: small RNA-seq: ago18HT-0.5mm-rep2


title: small RNA-seq: ago18HT-0.5mm-rep1


title: Drosophila testes, 48h PHS, Polysome fraction '60S', Replicate 2

description: biological replicate 2

value: 48 hours


title: embryo4_cell6

description: lane1_Ferdinand_Embryos_Plate2_C08_CAACCTCC


title: embryo4_cell5

description: lane1_Ferdinand_Embryos_Plate2_C07_CACATTGC


title: 35SLZ_WL_rep3

value: White light